GSM7712860: ArcPMX1; Mus musculus; RNA-Seq
Mouse microglial single-cell in hippocampi and hippocampi nuclei were collected for Split-seq and dispersed into 48 wells of a 96 well plate containing unique barcodes per well (24 wells of microglial cells, 24 wells of hippocampi nuclei). Half hippocampi from 2-3 mice of the same sex, genotype and treatment were pooled for microglia isolation and processing (HBSS with Actinomycin D (5 μg/ml), Triptolide (10 μM) and Anisomycin (27.1 μg/ml)) (3 samples per sex/genotype/treatment) .  Following dissociation with gentle MACS Octo Dissociator and Adult Brain Dissociation Kit, cells were filtered (70 µm), debris removed, and myelin was cleared with Myelin removal beads II.  Microglia were isolated using Anti-CD11b MicroBeads. Cells were counted and resuspended in 0.05% BSA in DPBS, to reach a minimum of 1,000 cells/µL up to around 7,000 cells/µL. For sn-RNA-Seq, perfused half hippocampi (3 per sex/genotype/treatment) were placed into Nuclei Extraction Buffer (Miltenyi) with 0.2 U/µL RNase inhibitor), processed in gentleMACSTM Octo Dissociator, filtered (70 µm), centrifuged and resuspended in PBS with 0.1% BSA and 0.2 U/µL RNase inhibitor, passed through a 30 µm filter, and counted). 4 million nuclei were collected from the stock of each cell preparation, and processed per Parse Biosciences. RNA was reverse transcribed in situ and barcoded cDNA was extracted following Parse Biosciences' Single Cell Whole Transcriptome Kit (also called Split-seq). Illumina Split-seq: Parse Biosciences' Single Cell Whole Transcriptome Kit (also called Split-seq). Library preparation was performed following manufacturer's instructions for the Single Cell Whole Transcriptome Kit, where the cells are mixed for the next 2 sets of barcoding reactions followed by sublibrary generation.